White Blood Cell Analysis System and Method

ABSTRACT

Systems and methods for analyzing blood samples, and more specifically for performing a white blood cell (WBC) differential analysis. The systems and methods screen WBCs by means of fluorescence staining and a fluorescence triggering strategy. As such, interference from unlysed red blood cells (RBCs) and fragments of lysed RBCs is substantially eliminated. The systems and methods also enable development of relatively milder WBC reagent(s), suitable for assays of samples containing fragile WBCs. In one embodiment, the systems and methods include: (a) staining a blood sample with an exclusive cell membrane permeable fluorescent dye, which corresponds in emission spectrum to an excitation source of a hematology instrument; (b) using a fluorescence trigger to screen the blood sample for WBCs; and (c) using measurements of (1) axial light loss, (2) intermediate angle scatter, (3) 90° polarized side scatter, (4) 90° depolarized side scatter, and (5) fluorescence emission to perform a differentiation analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 61/482,541, titled, Method ForAnalyzing White Blood Cells, and filed on May 4, 2011, the entiredisclosure of which is incorporated by reference herein.

This application is also related to Application No xx/xxx,xxx, filed onApr. 26, 2012, titled “NUCLEATED RED BLOOD CELL ANALYSIS SYSTEM ANDMETHOD,” with Atty Dkt No: ADDV-017 (11041US01); and application Noxx/xxx,xxx, filed on Apr. 26, 2012, titled “BASOPHIL ANALYSIS SYSTEM ANDMETHOD,” with Atty Dkt No: ADDV-018 (11042US01), the entire disclosuresof which are herein incorporated by reference in their entirety.

BACKGROUND

This invention relates to hematology systems and methods. Morespecifically, this invention relates to systems and methods foranalyzing blood samples to identifying, classify, and/or quantify whiteblood cells (WBC) and WBC sub-populations in a sample of blood.

The development of an accurate and efficient hematology assay foranalysis of WBCs, including counting and classification, has been achallenge. One reason for the difficulty in developing an accurate andefficient assay is the relatively low concentration of WBCs(approximately 0.1% to 0.2%) amongst total blood cells in a sample.Attempts to engineer advanced methods for analyzing WBCs, andformulating robust WBC reagent(s), have remained one of the toppriorities in the area of automated hematology analyzers.

Typically, lysis of red blood cells (RBCs) is required to eliminateinterference from RBCs, and concentrate WBCs, before counting andclassifying WBCs and WBC sub-populations. More specifically, accurateand efficient WBC analysis requires: (1) complete lysis of RBCs in lessthan 30 seconds; (2) the breaking of large fragments of RBCs intosmaller pieces after lysis; and (3) the preservation of WBCs foraccurate counting and proper classification. If the blood sample is“under-lysed,” unlysed RBCs, even in very small concentrations,interfere with WBC counting and differential analysis. Similarly, largerfragments of lysed RBCs can interfere with WBC counting and differentialanalysis. In practice, it is difficult to separate unlysed RBCs and/orlarger fragments of lysed RBCs from lymphocytes (the smallest WBCs). Ifthe blood sample is “over-lysed,” the classification of WBCs may beadversely affected on account of excessive damage to cell membranes ofWBCs.

In some instances, the difficulty of WBC analysis may be compounded bythe presence of two special types of samples; namely, samples containinglysis-resistant red blood cells (rstRBCs) and samples containing fragilelymphocytes. In the case of samples containing rstRBCs, the WBC countand percentage of lymphocytes are falsely reported “high,” on account ofthe contribution of particles other than true lymphocytes, consequentlyposing a risk of improper diagnoses and treatments for patients. In thecase of samples containing fragile lymphocytes, damaged lymphocytes maynot show their characteristics in a WBC differential analysis. Inaddition, exposed nuclei of WBCs may be counted as nucleated red bloodcells (nRBCs), resulting in a false positive count of nRBCs in certainassays.

BRIEF SUMMARY

Provided herein are systems and methods for analyzing blood samples, andmore specifically for performing a white blood cell (WBC) differentialanalysis. In general, the systems and methods disclosed screen WBCs bymeans of fluorescence staining and a fluorescence triggering strategy.As such, interference from unlysed RBCs (e.g., rstRBCs) and RBCfragments is substantially or completely eliminated, thereby ensuringaccurate counting and differentiation of WBCs and WBC sub-populations.The systems and methods also enable development of relatively milder WBCreagent(s), suitable for assays of samples containing fragilelymphocytes (or other fragile WBCs), including aged samples.

In one embodiment, for example, the systems and methods disclosed hereininclude: (a) staining a blood sample with an exclusive, cell membranepermeable, fluorescent dye, which corresponds in emission spectrum to anexcitation source of a hematology instrument; (b) using a fluorescencetrigger to screen the blood sample for WBCs; and (c) using a combinationof measurements of (1) axial light loss, (2) intermediate angle scatter,(3) 90° polarized side scatter, (4) 90° depolarized side scatter, and(5) fluorescence emission to perform a differential analysis.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated herein, form part ofthe specification. Together with this written description, the drawingsfurther serve to explain the principles of, and to enable a personskilled in the relevant art(s), to make and use the systems and methodspresented. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIGS. 1A-1E show histograms of a sample of whole blood, showing WBCs andresidues of RBCs following lysis.

FIG. 1A is a histogram showing the measurement of an axial light losssignal.

FIG. 1B is a histogram showing the measurement of intermediate anglescatter.

FIG. 1C is a histogram showing the measurement of 90° polarized sidescatter.

FIG. 1D is a histogram showing the measurement of 90° depolarized sidescatter.

FIG. 1E is a histogram showing the measurement of fluorescence.

FIG. 2 is a cytogram showing the use of a fluorescent trigger foreliminating any fragments of RBCs from consideration.

FIG. 3 is another cytogram showing the use of a fluorescent trigger foreliminating any fragments of RBCs from consideration.

FIG. 4 is a schematic diagram illustrating a hematology instrument.

FIGS. 5A-5J show cytograms of a five-part WBC differential analysis.

FIG. 5A is a cytogram depicting axial light loss vs. intermediate anglescatter.

FIG. 5B is a cytogram depicting 90° polarized side scatter vs. axiallight loss.

FIG. 5C is a cytogram depicting 90° polarized side scatter vs.intermediate angle scatter.

FIG. 5D is a cytogram depicting 90° depolarized side scatter vs. axiallight loss.

FIG. 5E is a cytogram depicting 90° depolarized side scatter vs.intermediate angle scatter.

FIG. 5F is a cytogram depicting 90° depolarized side scatter vs. 90°polarized side scatter.

FIG. 5G is a cytogram depicting fluorescence vs. axial light loss.

FIG. 5H is a cytogram depicting fluorescence vs. intermediate anglescatter.

FIG. 5I is a cytogram depicting fluorescence vs. 90° polarized sidescatter.

FIG. 5J is a cytogram depicting fluorescence vs. 90° depolarized sidescatter.

FIG. 6A is a cytogram illustrating analysis of a sample of whole bloodcontaining lyse-resistant RBCs, using a traditional method.

FIG. 6B is a cytogram showing axial light loss vs. intermediate anglescatter made by a fluorescence-triggered hematology analyzer, inaccordance with an embodiment presented.

FIG. 7A is a cytogram illustrating analysis of an aged sample of wholeblood, using a traditional method.

FIG. 7B is a cytogram showing axial light loss vs. intermediate anglescatter made by a fluorescence-triggered fluorescence-triggeredhematology analyzer, in accordance with an embodiment presented.

DETAILED DESCRIPTION

Provided herein are systems and methods for analyzing blood samples, andmore specifically for performing a white blood cell (WBC) differentialanalysis to identify, classify, and count WBCs and WBC sub-populations.In general, the systems and methods disclosed screen WBCs by means offluorescence staining and a fluorescence triggering strategy. As such,interference from unlysed red blood cells (RBCs), such aslysis-resistant red blood cells (rstRBCs), and RBC fragments issubstantially eliminated. The systems and methods disclosed therebyensure accurate counting and differentiation of WBCs and WBCsub-populations. The systems and methods also enable development ofrelatively milder WBC reagent(s), suitable for assays of samplescontaining fragile lymphocytes (or other fragile WBCs), including agedsamples.

In one embodiment, for example, the systems and methods disclosed hereininclude: (a) staining a blood sample with an exclusive, cell membranepermeable, fluorescent dye, which corresponds in emission spectrum to anexcitation source of a hematology instrument; (b) using a fluorescencetrigger to screen the blood sample for WBCs; and (c) using a combinationof measurements of (1) axial light loss, (2) intermediate angle scatter,(3) 90° polarized side scatter, (4) 90° depolarized side scatter, and(5) fluorescence emission to perform a WBC differential analysis.

As used herein, the expression “fluorescence information” means datacollected from a fluorescence channel of a hematology analyzer. As usedherein, the expression “fluorescence channel” means a detection device,such as a photomultiplier tube, set at an appropriate wavelength bandfor measuring the quantity of fluorescence emitted from a sample.

(1) Use of Fluorescent Dye(s).

WBCs contain a relatively high concentration of DNA in their nuclei.Mature RBCs, however, do not contain DNA. Therefore, a fluorescent dyeis selected to differentiate two classes of blood cells; namely, theblood cells containing nucleic acids and the blood cells not containingnucleic acids. The purpose of the dye is to penetrate into live cellseasily, bind DNA with high affinity, and emit strong fluorescence withadequate Stokes shift when the dye is excited by an appropriate sourceof light. The peak absorption of the dye in the visible bandsubstantially matches the wavelength of the source of light (within 50nm of the wavelength of the source of light, more preferably, within 25nm of the wavelength of the source of light), in order to be properlyexcite the dye and achieve optimal results.

The fluorescent dye selected is preferably: 1) capable of bindingnucleic acids, 2) capable of penetrating cell membranes of WBCs, 3)excitable at a selected wavelength when subjected to a source of light,4) emits fluorescence upon excitation by the source of light, and 5) isbiostable and soluble in a liquid. The dye may be selected from groupconsisting of: acridine orange, SYBR 11, SYBR Green series dye, hexidiumiodide, SYTO 11, SYTO 12, SYTO 13, SYTO 14, SYTO 16, SYTO 21, SYTO RNASelect, SYTO 24, SYTO 25 and any equivalents thereof. The dye is used to“activate” WBCs and “screen out” unlysed RBCs and fragments of RBCsbased on a fluorescence trigger configured in the hematology analyzer.The dye is typically present at a concentration of from about 0.1 ng/mLto about 0.1 mg/mL. While various dyes are available, the dye selectedis generally paired with the excitation source of the hematologyanalyzer such that a single exclusive dye is used to stain and excitefluorescence emission in all WBC sub-populations intended to beidentified, quantified, and/or analyzed. As such, a single (i.e.,exclusive) dye can be used to identify, quantify, and analyze all theWBC subpopulations at once.

In one embodiment, a fluorescent dye is provided in a WBC reagent, withcombinations of 1) at least one surfactant, 2) at least one buffer, 3)at least one salt, and/or 4) at least antimicrobial agent, in sufficientquantities for carrying out staining and activating up to 1,000×10³ WBCsper microliter. The at least one surfactant, such as “TRITON” X-100 orsaponin, is used to destroy the membranes of RBC, and reduce the sizesof fragments of RBCs. The at least one surfactant is typically presentat a concentration of from about 0.001% to about 5%. The at least oneantimicrobial agent, such as those from “TRIADINE” or “PROCLIN”families, is used to prevent the contamination of the reagent frommicrobes. The concentration of the at least one antimicrobial agent issufficient to preserve the reagent for the shelf life required. The atleast one buffer, such as phosphate buffered saline (PBS) or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), is used toadjust the pH of reaction mixture for controlling lysis of RBCs andpreserving WBCs. The at least one buffer is typically present at aconcentration of from about 0.01% to about 3%. The pH typically rangesfrom about 3 to about 12. The at least one salt, such as NaCl or Na₂SO₄,is used to adjust the osmolality to increase the effect of lysing and/oroptimize WBC preservation. The at least one salt may be present at aconcentration of from about 0.01% to about 3%. In certain cases, the atleast one buffer can serve as the at least one salt, or the at least onesalt can serve as the at least one buffer. In general, lower osmolality,or hypotonicity, is used to accelerate the lysis of RBCs. The osmolalitytypically ranges from about 20 to about 250 mOsm.

Lysis of RBCs can be made to occur at a temperature above roomtemperature (e.g., between about 30° C. to about 50° C., such as about40° C.) over a relatively short period of time (e.g., less than about 25seconds, less than about 17 seconds, or even less than about 9 seconds),following mixing of the sample of blood and the WBC reagent at a ratioof about one part by volume sample to about 35 parts by volume WBCreagent. The data for analysis is collected with a plurality of opticalchannels and at least one fluorescence channel.

FIGS. 1A-E show the separation of true WBCs from unlysed RBCs and RBCfragments, in histograms of collected optical information and ahistogram of fluorescence information. The histogram in FIG. 1A shows ameasurement of axial light loss (ALL). The histogram in FIG. 1B shows ameasurement of intermediate angle scatter (IAS). The histogram in FIG.1C shows a measurement of 90° polarized side scatter (PSS). Thehistogram in FIG. 1D shows a measurement of 90° depolarized side scatter(DSS). The histogram in FIG. 1E shows a measurement of fluorescence(FL1). In the histograms, the horizontal axis indicates the value of thedetection channel (or the names of the channels, i.e., ALL, IAS, PSS,DSS or FL1). The vertical axis indicates counts of components of thesample of blood. In the histograms, the lines 100 indicate residues ofRBCs and lines 200 indicate WBCs. As used herein, “residues of RBCs” issynonymous with “fragments of RBCs.” As shown by comparing FIG. 1E toFIGS. 1A-1D, fluorescence information shows much better separationbetween the two groups of particles (i.e., WBCs and residues of RBCs)than do any of the optical channels, thereby facilitating the followinganalysis.

(2) Use of a Fluorescence Trigger.

Blood cells emit different magnitudes of fluorescence signals uponexcitation of the fluorescent dye by a source of light. The differencesin magnitude of fluorescence signals arise from the quantity of nucleicacids, namely DNA, inside the cells. The greater the quantity of DNA,the greater the likelihood of higher fluorescence signals. Also,efficacy of penetration of cell membranes, size of the dye, bindingkinetics between the dye and DNA, affinity between the dye and DNA, andother factors, affect the fluorescence signals. Mature RBCs emit minimalfluorescence signals because there is no DNA within mature RBCs.Nucleated red blood cells (nRBCs) emit very strong fluorescence signals,because not only is DNA inside nuclei of nRBCs, but also the staining iseasier because membranes of nRBCs are destroyed during the lysisprocedure. Unlysed RBCs or RBC fragments do not emit fluorescence,although they may emit very weak auto-fluorescence. As shown withreference to FIG. 1E, the cells that emit much stronger fluorescencesignals are the cells having nuclei, namely, WBCs (and nRBCs whenpresent).

As such, the systems and methods presented herein use a fluorescencetrigger for collecting and analyzing WBCs. For example, a fluorescencetrigger, usually set between signals from RBCs and signals from WBCs,can be used to collect signals from WBCs separately for furtheranalysis. Two examples of using an FL1 trigger are shown in FIG. 2 andFIG. 3. FIG. 2 is a cytogram showing the use of a fluorescent triggerfor eliminating any fragments of RBCs (nuclei-free particles) andcollecting nuclei-containing events (e.g., WBCs and/or nRBCs). Thefluorescent dye was acridine orange and the concentration of thefluorescent dye was 3 μg/mL. The voltage of the fluorescentphotomultiplier tube was set at 350 volts. FIG. 3 is a cytogram showingthe use of a fluorescent trigger for eliminating any fragments of RBCs(nuclei-free particles) and collecting nuclei-containing events (e.g.,WBCs and/or nRBCs). The fluorescent dye was acridine orange and theconcentration of the fluorescent dye was 0.03 μg/mL. The voltage of thefluorescent photomultiplier tube was set at 500 volts. In a WBC assayusing acridine orange staining (even with drastically differentconcentrations of the dyes, i.e., 3 μg/mL in FIGS. 2 and 0.03 μg/mL inFIG. 3) and a properly set FL1 trigger, only the events above the FL1trigger are nuclei-containing events (e.g., WBCs and/or nRBCs, ifpresent) and, consequently, are captured for further analysis.

(3) Use of a Plurality of Optical Channels and at Least One FluorescenceChannel for Analysis.

In one embodiment, the WBC differential analysis is conducted by meansof Multiple Angle Polarized Scattering Separation technology (MAPSS),with enhancement from fluorescence information. At least one photodiode,or at least one photomultiplier tube, or both at least one photodiodeand at least one photomultiplier tube, are needed to detect lightscattered by each blood cell passing through a flow cell. Two or morephotodiodes are used for measuring ALL signals, which measure about 0°scatter, and IAS signals, which measure low angle (e.g., about 3° toabout 15°) scatter. Two or more photomultiplier tubes are used fordetecting 90° PSS signals and 90° DSS signals. Additionalphotomultiplier tubes are needed for FL1 measurements within appropriatewavelength range(s), depending on the choice of wavelength of the sourceof light. Each event captured on the system thus exhibits a plurality ofdimensions of information, such as ALL, IAS (one or more channels), PSS,DSS, and fluorescence (one or more channels). The information from thesedetection channels is used for further analysis of blood cells.

FIG. 4 is a schematic diagram illustrating the illumination anddetection optics of an apparatus suitable for hematology analysis(including flow cytometry). Referring now to FIG. 4, an apparatus 10comprises a source of light 12, a front mirror 14 and a rear mirror 16for beam bending, a beam expander module 18 containing a firstcylindrical lens 20 and a second cylindrical lens 22, a focusing lens24, a fine beam adjuster 26, a flow cell 28, a forward scatter lens 30,a bulls-eye detector 32, a first photomultiplier tube 34, a secondphotomultiplier tube 36, and a third photomultiplier tube 38. Thebulls-eye detector 32 has an inner detector 32 a for 0° light scatterand an outer detector 32 b for 7° light scatter.

In the discussion that follows, the source of light is preferably alaser. However, other sources of light can be used, such as, forexample, lamps (e.g., mercury, xenon). The source of light 12 can be avertically polarized air-cooled Coherent Cube laser, commerciallyavailable from Coherent, Inc., Santa Clara, Calif. Lasers havingwavelengths ranging from 350 nm to 700 nm can be used. Operatingconditions for the laser are substantially similar to those of laserscurrently used with “CELL-DYN” automated hematology analyzers.

Additional details relating to the flow cell, the lenses, the focusinglens, the fine-beam adjust mechanism and the laser focusing lens can befound in U.S. Pat. No. 5,631,165, incorporated herein by reference,particularly at column 41, line 32 through column 43, line 11. Theforward optical path system shown in FIG. 4 includes a sphericalplano-convex lens 30 and a two-element photo-diode detector 32 locatedin the back focal plane of the lens. In this configuration, each pointwithin the two-element photodiode detector 32 maps to a specificcollection angle of light from cells moving through the flow cell 28.The detector 32 can be a bulls-eye detector capable of detecting axiallight loss (ALL) and intermediate angle forward scatter (IAS). U.S. Pat.No. 5,631,165 describes various alternatives to this detector at column43, lines 12-52.

The first photomultiplier tube 34 (PMT1) measures depolarized sidescatter (DSS). The second photomultiplier tube 36 (PMT2) measurespolarized side scatter (PSS), and the third photomultiplier tube 38(PMTS) measures fluorescence emission from 440 nm to 680 nm, dependingupon the fluorescent dye selected and the source of light employed. Thephotomultiplier tube collects fluorescent signals in a broad range ofwavelengths in order to increase the strength of the signal.Side-scatter and fluorescent emissions are directed to thesephotomultiplier tubes by dichroic beam splitters 40 and 42, whichtransmit and reflect efficiently at the required wavelengths to enableefficient detection. U.S. Pat. No. 5,631,165 describes variousadditional details relating to the photomultiplier tubes at column 43,line 53 though column 44, line 4.

Sensitivity is enhanced at photomultiplier tubes 34, 36, and 38, whenmeasuring fluorescence, by using an immersion collection system. Theimmersion collection system is one that optically couples the first lens30 to the flow cell 28 by means of a refractive index matching layer,enabling collection of light over a wide angle. U.S. Pat. No. 5,631,165describes various additional details of this optical system at column44, lines 5-31.

The condenser 44 is an optical lens system with aberration correctionsufficient for diffraction limited imaging used in high resolutionmicroscopy. U.S. Pat. No. 5,631,165 describes various additional detailsof this optical system at column 44, lines 32-60.

The functions of other components shown in FIG. 4, i.e., a slit 46, afield lens 48, and a second slit 50, are described in U.S. Pat. No.5,631,165, at column 44, line 63 through column 45, line 26. Opticalfilters 52 or 56 and a polarizer 52 or 56, which are inserted into thelight paths of the photomultiplier tubes to change the wavelength or thepolarization or both the wavelength and the polarization of the detectedlight, are also described in U.S. Pat. No. 5,631,165, at column 44, line63 through column 45, line 26. Optical filters that are suitable for useherein include band-pass filters and long-pass filters.

The photomultiplier tubes 34, 36, and 38 detect either side-scatter(light scattered in a cone whose axis is approximately perpendicular tothe incident laser beam) or fluorescence (light emitted from the cellsat a different wavelength from that of the incident laser beam).

While select portions of U.S. Pat. No. 5,631,165 are referenced above,U.S. Pat. No. 5,631,165 is incorporated herein by reference in itsentirety.

FIGS. 5A-J show an example of an enhanced five-part WBC differentialanalysis. Neutrophils (NE), lymphocytes (LY), monocytes (MO),eosinophils (EO) and basophils (BA) were separated using MAPSStechnology and a FL1 channel. The cytogram in FIG. 5A shows ALL vs. IAS.The cytogram in FIG. 5B shows 90° PSS vs. ALL. The cytogram in FIG. 5Cshows 90° PSS vs. IAS. The cytogram in FIG. 5D shows 90° DSS vs. ALL.The cytogram in FIG. 5E shows 90° DSS vs. IAS. The cytogram in FIG. 5Fshows 90° DSS vs. 90° PSS. The cytogram in FIG. 5G shows FL1 vs. ALL.The cytogram in FIG. 5H shows FL1 vs. IAS. The cytogram in FIG. 5I showsFL1 vs. 90° PSS. The cytogram in FIG. 5J shows FL1 vs. 90° DSS.

In addition to the information collected from the four traditional MAPSSchannels (ALL, IAS, PSS, DSS), the FL1 channel further distinguishes thecell sub-populations (FIGS. 5G through 5J, inclusive). For the case inwhich acridine orange is used as the dye for screening WBCs, basophilsshow relatively low FL1 signals, and monocytes show relatively high FL1signals, relative to other WBC sub-populations, i.e., neutrophils,eosinophils, and lymphocytes. In the cytograms, dots 510 representneutrophils, dots 520 represent eosinophils, dots 530 representlymphocytes, dots 540 represent basophils, and dots 550 representmonocytes. The combined quantitative information from all opticaldimensions and the fluorescence dimension provides an enhanced, and morereliable, differential analysis for samples of blood containing WBCs.

FIG. 6A is a cytogram illustrating analysis of a sample of whole bloodcontaining rstRBCs, using a traditional method. FIG. 6A shows a cytogramof ALL vs. IAS, using a commercially available “CELL-DYN” Sapphire™hematology analyzer. FIG. 6B is a cytogram showing ALL vs. IAS, using anFL1 trigger-enhanced hematology analyzer. The sample of whole blood wasthe same as that analyzed in FIG. 6A. The concentration of thefluorescent dye, acridine orange, was 3 μg/mL. The results show that themethod described herein is accurate and efficient for the two mostchallenging cases mentioned previously. In the traditional method,unlysed RBCs, i.e., the events appearing in the lower left of thecytogram, were recognized as lymphocytes, thereby resulting in a highercount of WBCs and a higher percentage of lymphocytes. FIG. 6B shows theresults of an analysis of WBCs of the sample containing rstRBCs by themethod described herein. In the method described herein, the analysis ofWBCs was accurate because no RBCs or residues of RBCs were recognized.

FIG. 7A is a cytogram illustrating analysis of an aged (28 hours old)sample of whole blood, which contains more fragile WBCs, using atraditional method. FIG. 7A is a cytogram showing ALL vs. IAS, using a“CELL-DYN” Sapphire™ hematology analyzer. FIG. 7B is a cytogram showingALL vs. IAS, using an FL1 trigger-enhanced hematology analyzer. Thesample of whole blood was the same as that analyzed in FIG. 7A. Theconcentration of the fluorescent dye, acridine orange, was 3 μg/mL.

The methods described herein enhances WBC analysis for hematologyanalyzers. The methods described herein provides a more accurate WBCcount and a more accurate classification of WBC sub-populations, becausethe interference from unlysed RBCs and RBC fragments is substantiallyeliminated. The use of fluorescence provides further information toimprove differential analysis of WBCs. The methods described hereinshows advantages over traditional methods when analyzing samples havingrstRBCs and samples having fragile WBCs.

Additional Embodiments

In one embodiment, there is provided a hematology analyzer forconducting a WBC differential analysis on a blood sample that has beendyed with a fluorescent dye. The analyzer comprises an excitation sourcepositioned to excite particles within the blood sample. The analyzerfurther comprises a plurality of detectors including: (1) an axial lightloss detector positioned to measure axial light loss from the excitedblood sample, (2) an intermediate angle scatter detector positioned tomeasure intermediate angle scatter from the excited blood sample, (3) apolarized side scatter detector positioned to measure 90° polarized sidescatter from the excited blood sample, (4) a depolarized side scatterdetector positioned to measure 90° depolarized side scatter from theexcited blood sample, and (5) a fluorescence detector positioned tomeasure fluorescence emitted from the excited blood sample. The analyzerfurther comprises a processor configured to receive the measurements of(1) axial light loss, (2) intermediate angle scatter, (3) 90° polarizedside scatter, (4) 90° depolarized side scatter, and (5) fluorescencefrom the plurality of detectors. The processor is also configured toperform a WBC differential analysis of the blood sample, based on allfive measurements, for particles that emit fluorescence above afluorescence threshold. The processor may be further configured topre-screen the received measurements to remove from consideration anyparticles that do not meet the fluorescence threshold. The axial lightloss detector may measure axial light loss at 0° scatter. Theintermediate angle scatter detector may measure light angle scatter atabout 3° to about 15°. The plurality of detectors may include one ormore photomultiplier tubes. The excitation source may be a laserconfigured to emit light at a wavelength corresponding to thefluorescent dye. Alternatively, the fluorescent dye may be selected tocorrespond with the excitation source. The fluorescent dye may be cellmembrane permeable, and nucleic acid binding.

The hematology analyzer may further comprise an incubation subsystem fordiluting the blood sample with a WBC reagent. The WBC reagent mayinclude the fluorescent dye and one or more lysing agents.Alternatively, the WBC reagent may include (a) at least one surfactant,(b) at least one buffer or at least one salt, (c) at least oneantimicrobial agent, and (d) the fluorescent dye. The incubationsubsystem may be configured to incubate the blood sample with the WBCreagent for a period of time of less than about 25 seconds, less thanabout 17 seconds, or less than about 9 seconds. The incubation subsystemmay also be configured to incubate the blood sample with the WBC reagentat a temperature ranging from about 30° C. to about 50° C., such asabout 40° C.

In another embodiment, there is provided a method of configuring ahematology analyzer to perform a WBC differential analysis on a bloodsample that has been dyed with a fluorescent dye. The method includespositioning an excitation source to excite particles within the bloodsample. The method further includes positioning a plurality of detectorsto measure (1) axial light loss, (2) intermediate angle scatter, (3) 90°polarized side scatter, (4) 90° depolarized side scatter, and (5)fluorescence from the excited blood sample. The method further comprisesconfiguring a processor configured to receive the measurements of (1)axial light loss, (2) intermediate angle scatter, (3) 90° polarized sidescatter, (4) 90° depolarized side scatter, and (5) fluorescence from theplurality of detectors. The method also includes configuring theprocessor to perform a WBC differential analysis of the blood sample,based on all five measurements, for particles that emit fluorescenceabove a fluorescence threshold. The method may include configuring theprocessor to pre-screen the received measurements to remove fromconsideration any particles that do not meet the fluorescence threshold.The axial light loss detector may measure axial light loss at 0°scatter. The intermediate angle scatter detector may measure light anglescatter at about 3° to about 15°. The plurality of detectors may includeone or more photomultiplier tubes. The method may also includeconfiguring the excitation source to emit light at a wavelengthcorresponding to the fluorescent dye. Alternatively, the fluorescent dyemay be selected to correspond with the excitation source. Thefluorescent dye may be cell membrane permeable, and nucleic acidbinding.

The method may further comprise configuring an incubation subsystem ofthe hematology analyzer to incubate the blood sample with a WBC reagent.The WBC reagent may include the fluorescent dye and a lysing agent.Alternatively, the WBC reagent may include (a) at least one surfactant,(b) at least one buffer or at least one salt, (c) at least oneantimicrobial agent, and (d) the fluorescent dye. The incubationsubsystem may be configured to incubate the blood sample with the WBCreagent for a period of time of less than about 25 seconds, less thanabout 17 seconds, or less than about 9 seconds. The incubation subsystemmay also be configured to incubate the blood sample with the WBC reagentat a temperature ranging from about 30° C. to about 50° C., such asabout 40° C.

In another embodiment, there is provided a hematology analyzer forperforming a WBC differential analysis, comprising: (1) means forexciting particles within a blood sample, which includes positioning alaser light source to excite the blood sample as it transverses aflowcell of the hematology analyzer, or equivalents thereof; (2) meansfor measuring a plurality of light scatter signals from the excitedparticles within the blood sample, which includes a plurality ofdetectors (as discussed above), or equivalents thereof; (3) means formeasuring a fluorescence signal from the excited particles within theblood sample, which includes fluorescence detectors (as discussedabove), or equivalents thereof; (4) means for screening the excitedparticles to remove from consideration any particles that do not meet afluorescence threshold, which includes a processor configured with afluorescence trigger (as discussed above), or equivalents thereof; and(5) means for performing a WBC differential analysis, based on theplurality of light scatter signals and the fluorescence signal, for theparticles passing the means for screening, which includes a processorconfigured to perform a WBC differential (as discussed above), orequivalents thereof. The hematology analyzer may further comprise meansfor incubating the blood sample with a WBC reagent for an incubationperiod of less than about 25 seconds, which includes an incubationsubsystem (as discussed above), or equivalents thereof. The hematologyanalyzer may further comprise means for incubating the blood sample witha WBC reagent at a temperature ranging from about 30° C. to about 50°C., which includes an incubation subsystem (as discussed above), orequivalents thereof.

In another embodiment, there is provided a method of performing a WBCanalysis with an automated hematology analyzer. The method comprises:(a) diluting a sample of whole blood with a WBC reagent, wherein the WBCreagent includes a RBCs lysing agent and a fluorescent dye thatpenetrates WBC membranes and binds to WBC nucleic acids; (b) incubatingthe diluted blood sample of step (a) for an incubation period of lessthan about 25 seconds, at a temperature ranging from about 30° C. toabout 50° C.; (c) delivering the incubated sample from step (b) to aflow cell in the hematology analyzer; (d) exciting the incubated samplefrom step (c) with an excitation source as the incubated sampletraverses the flow cell; (e) collecting a plurality of light scattersignals and a fluorescence emission signal from the excited sample; and(f) performing a WBC differential based on all the signals collected instep (e), while removing from consideration any particles within thediluted blood sample that do not meet a fluorescence threshold based onthe fluorescence emission signal. The WBC reagent may include (a) atleast one surfactant, (b) at least one buffer or at least one salt, (c)at least one antimicrobial agent, and (d) at least one fluorescent dye.The excitation source may have a wavelength of from about 350 nm toabout 700 nm. The fluorescence emission may be collected at a wavelengthof from about 360 nm to about 750 nm, by a band-pass filter or along-pass filter.

In yet another embodiment, there is provided a method for counting andclassifying WBCs by means of an automated hematology analyzer, themethod comprising the steps of: (a) diluting a sample of whole bloodwith at least one WBC reagent; (b) incubating the diluted sample of step(a) for sufficient period of time within a selected temperature range tolyse RBCs, to preserve WBCs, to allow at least one fluorescent dye topenetrate cell membranes of the WBCs, and bind nucleic acids within thenuclei of the WBCs; (c) delivering the incubated sample from step (b) toa flow cell in a stream; (d) exciting the incubated sample from step (c)by means of a source of light as the incubated sample traverses the flowcell; (e) collecting a plurality of optical scatter signals and at leastone fluorescence emission signal simultaneously; and (f) differentiatingand quantifying WBCs by means of the signals collected in step (e).Features of the embodiment include, but are not limited to: (1) use ofat least one fluorescent dye to bind and stain nucleic acids in WBCs andother nuclei-containing cells in a given sample of blood during theprocedure for lysing RBCs, and to induce fluorescent emissions uponexcitation by photons from a given source of light, such as a laser beamat an appropriate wavelength; (2) use of a fluorescent trigger toseparate and collect events that emit strong fluorescence (e.g., eventsinvolving WBCs and other nuclei-containing cells); (3) use of aplurality of optical channels and at least one channel for fluorescencefor collecting data and analyzing the data so collected in order toidentify each cell population and reveal additional information.

In yet another embodiment, the systems and methods disclosed hereininclude: (a) staining a blood sample with an exclusive, cell membranepermeable, fluorescent dye, which corresponds in emission spectrum to anexcitation source of a hematology instrument; (b) using a fluorescencetrigger to screen the blood sample for WBCs; and (c) using a combinationof measurements to perform a differential analysis. The combination ofmeasurements may include one or more measurements selected from thegroup consisting of: axial light loss, intermediate angle scatter, 90°polarized side scatter, 90° depolarized side scatter, one or morefluorescence emission measurements, multiple ring intermediate anglescatter, and any combinations or equivalents thereof.

In one embodiment, the invention is directed toward one or more computersystems capable of carrying out the functionality described herein. Forexample, any of the method/analysis steps discussed herein may beimplemented in a computer system having one or more processors, a datacommunication infrastructure (e.g., a communications bus, cross-overbar, or network), a display interface, and/or a storage or memory unit.The storage or memory unit may include computer-readable storage mediumwith instructions (e.g., control logic or software) that, when executed,cause the processor(s) to perform one or more of the functions describedherein. The terms “computer-readable storage medium,” “computer programmedium,” and “computer usable medium” are used to generally refer tomedia such as a removable storage drive, removable storage units, datatransmitted via a communications interface, and/or a hard disk installedin a hard disk drive. Such computer program products provide computersoftware, instructions, and/or data to a computer system, which alsoserve to transform the computer system from a general purpose computerinto a special purpose computer programmed to perform the particularfunctions described herein. Where appropriate, the processor, associatedcomponents, and equivalent systems and sub-systems thus serve asexamples of “means for” performing select operations and functions. Such“means for” performing select operations and functions also serve totransform a general purpose computer into a special purpose computerprogrammed to perform said select operations and functions.

CONCLUSION

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed.Other modifications and variations may be possible in light of the aboveteachings. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,and to thereby enable others skilled in the art to best utilize theinvention in various embodiments and various modifications as are suitedto the particular use contemplated. It is intended that the appendedclaims be construed to include other alternative embodiments of theinvention; including equivalent structures, components, methods, andmeans.

The above Detailed Description refers to the accompanying drawings thatillustrate one or more exemplary embodiments. Other embodiments arepossible. Modifications may be made to the embodiment described withoutdeparting from the spirit and scope of the present invention. Therefore,the Detailed Description is not meant to be limiting. Further, theSummary and Abstract sections may set forth one or more, but not allexemplary embodiments of the present invention as contemplated by theinventor(s), and thus, are not intended to limit the present inventionand the appended claims in any way.

1-19. (canceled)
 20. A method of performing a white blood cell (WBC)analysis with an automated hematology analyzer, the method comprising:(a) staining a sample of whole blood with a fluorescent dye thatpenetrates WBC membranes and binds to WBC nucleic acids; (b) excitingthe sample from step (a) with an excitation source as the sampletraverses a flow cell in the hematology analyzer; (c) collecting aplurality of light scatter signals and a fluorescence emission signalfrom the excited sample; (d) prior to performing a WBC differentialanalysis, excluding nuclei-free particles and retainingnuclei-containing particles using only a fluorescence trigger configuredin the hematology analyzer and that is limited to fluorescence emissionsignals and is set to a fluorescence magnitude that is greater thanfluorescence emission signals from RBCs, including RBC fragments, and isless than fluorescence emission signals from WBCs; and (e) performingthe WBC differential on the nuclei-containing particles retained in step(d).
 21. (canceled)
 22. The method of claim 20, wherein the excitationsource has a wavelength of from about 350 nm to about 700 nm.
 23. Themethod of claim 20, wherein fluorescence emission is collected at awavelength of from about 360 nm to about 750 nm, by a band-pass filteror a long-pass filter.
 24. The method according to claim 20, wherein thenuclei-containing events comprise signals from WBCs.
 25. The methodaccording to claim 20, wherein the nuclei-containing events comprisesignals from nucleated red blood cells (nRBCs).
 26. The method accordingto claim 20, wherein performing the WBC differential comprisesdistinguishing the nuclei-containing events from one another based onthe magnitude of the fluorescence emission signal associated with eachevent.
 27. The method according to claim 20, wherein performing the WBCdifferential comprises distinguishing the nuclei-containing events fromone another based on the plurality of light scatter signals associatedwith each event.
 28. The method according to claim 20, wherein theplurality of light scatter signals includes axial light loss (ALL)signals.
 29. The method according to claim 28, wherein the ALL signalsare measured at 0° scatter.
 30. The method according to claim 20,wherein the plurality of light scatter signals includes intermediateangle scatter (IAS) signals.
 31. The method according to claim 30,wherein the IAS signals are measured at about 3° to about 15° scatter.32. The method according to claim 20, wherein the plurality of lightscatter signals includes polarized side scatter (PSS) signals.
 33. Themethod according to claim 32, wherein the PSS signals are measured atabout 90° to scatter.
 34. The method according to claim 20, wherein theplurality of light scatter signals includes depolarized side scatter(DSS) signals.
 35. The method according to claim 34, wherein the DSSsignals are measured at about 90° to scatter.
 36. The method accordingto claim 20, further comprising incubating the blood sample of step (a)for an incubation period of time.
 37. The method according to claim 36,wherein the incubation period of time is less than 25 seconds.
 38. Themethod according to claim 36, wherein the incubation period of time isless than 17 seconds.
 39. The method according to claim 36, wherein theincubation period of time is less than 9 seconds.
 40. The methodaccording to claim 36, wherein the blood sample is incubated at atemperature ranging from 30° C. to 50° C.
 41. The method according toclaim 36, wherein the blood sample is incubated at a temperature ofabout 40° C.
 42. The method according to claim 20, wherein a singlefluorescent dye is used to identify, quantify, and analyze a pluralityof WBC subpopulations at once.